Thrombomodulin is known as a substance having an action of specifically binding to thrombin to inhibit the blood coagulation activity of thrombin, and at the same time, significantly promote the ability of thrombin to activate Protein C, and is also known to have strong blood coagulation-inhibiting action. It is also known that thrombomodulin extends the thrombin clotting time, and that it suppresses platelet aggregation by thrombin. Protein C is a vitamin K-dependent protein that plays an important role in the blood coagulation fibrinolytic system, and is activated by the action of thrombin to become activated Protein C. It is known that the activated Protein C inactivates activated blood coagulation factor V and activated blood coagulation factor VIII in vivo, and that it is involved in generation of plasminogen activator having thrombolytic action (Non-patent document 1). Therefore, thrombomodulin is considered to be useful as an anticoagulant agent or a thrombolytic agent that promotes the activation of Protein C by thrombin, and there have also been reported animal experiments demonstrating that thrombomodulin is effective for therapeutic and prophylactic treatments of diseases associated with acceleration of coagulation (Non-patent document 2).
Thrombomodulin was first discovered and obtained as a glycoprotein expressed on vascular endothelial cells of various animal species including human, and thereafter successfully cloned. More specifically, a human thrombomodulin precursor gene containing a signal peptide was cloned from a human lung cDNA library by genetic engineering techniques, and the entire gene sequence of thrombomodulin was analyzed, so that the amino acid sequence consisting of 575 residues containing a signal peptide (usually 18 amino acid residues are exemplified) was elucidated (Patent document 1). It is known that mature thrombomodulin, from which the signal peptide has been cleaved, is constituted by 5 regions, namely, an N-terminal region (amino acids 1 to 226, these positions are indicated on the assumption that the signal peptide consists of 18 amino acid residues, and the same shall apply to the other regions), a region having six EGF-like structures (amino acids 227 to 462), an O-linked glycosylation region (amino acids 463 to 498), a transmembrane region (amino acids 499 to 521), and an intracytoplasmic region (amino acids 522 to 557), from the N-terminal side of the mature peptide, and that a part having the same activity as that of the full length thrombomodulin (i.e., minimum unit for the activity) mainly consists of the 4th, 5th, and 6th EGF-like structure portions from the N-terminal side among the six EGF-like structures (Non-patent document 3).
Unless a surfactant is present, the full length thrombomodulin is hardly dissolved, and therefore addition of a surfactant is essential for producing a thrombomodulin preparation. In contrast, there is also soluble thrombomodulin that can be fully dissolved even in the absence of a surfactant. The soluble thrombomodulin may be prepared so as not to contain at least a part of the transmembrane region or the entire transmembrane region. For example, it has been confirmed that a soluble thrombomodulin consisting of only 3 regions of the N-terminal region, the region having six EGF-like structures, and the O-linked glycosylation region (i.e., soluble thrombomodulin having an amino acid sequence comprising amino acids at the positions 19 to 516 in SEQ ID NO: 1), can be obtained by applying recombination techniques, and that such recombinant soluble thrombomodulin has the same activity as that of the natural thrombomodulin (Patent document 1). In addition, there are also some other reports regarding soluble thrombomodulin (Patent documents 2 to 9). Further, human urine-derived soluble thrombomodulin and the like are also exemplified as natural thrombomodulin (Patent documents 10 and 11).
As recognized in many cases, as a result of spontaneous mutations or mutations occurring at the time of obtaining thrombomodulin, polymorphic mutations have been found even in human genes, and at present, such thrombomodulin genes that the amino acid at the position 473 of the human thrombomodulin precursor, that has the aforementioned amino acid sequence consisting of 575 amino acid residues, is Val or Ala have been identified. This difference corresponds to the difference of the nucleotide at the position 1418 to T or C in the nucleotide sequences encoding the amino acid (Non-patent document 4). However, these two thrombomodulins are completely identical in terms of their activities and physical properties. Thus, it can be considered that they are substantially identical.
It has been reported that thrombomodulin is effective for a therapeutic treatment of DIC (Non-patent documents 5 and 6). As for use of thrombomodulin, in addition to the aforementioned uses, thrombomodulin is expected to be used in therapeutic and prophylactic treatments of various diseases such as acute coronary syndrome (ACS), thrombosis, peripheral vessel obstruction, obstructive arteriosclerosis, vasculitis, functional disorder occurring after heart surgery, complication caused by organ transplantation, angina pectoris, transient ischemic attack, toxemia of pregnancy, diabetes, liver VOD (liver veno-occlusive disease, e.g., fulminant hepatitis, veno occlusive disease of liver occurring after bone marrow transplantation), and deep venous thrombosis (DVT), and further, adult respiratory distress syndrome (ARDS).
As a premise of application of thrombomodulin in pharmaceutical products, it is needless to explain that the soluble thrombomodulin is required to be manufactured in a large scale and at a cost as low as possible. However, there is also pointed out a possibility that heterogenous proteins originated in the production process, for example, proteins originated in host cells, bovine serum proteins originated in medium, and mouse IgG and the like originated in antibody column serve as immunogens to case problems concerning safety (Non-patent document 7).
As methods for producing soluble thrombomodulin in an industrial scale for application as a pharmaceutical product, there are known, for example, a method of using affinity column chromatography in a main purification step to which an antibody that reacts with thrombomodulin is bound, a method for producing highly purified soluble thrombomodulin substantially free from serum-originated substances and antibody-originated substances, which is characterized in that the soluble thrombomodulin is obtained as a flow-through fraction in a step of bringing the soluble thrombomodulin obtained by affinity column chromatography into contact with a cation exchanger under conditions of a specific conductivity of 25 to 34 ms/cm and pH 3 to 4 (Patent document 12), and a method for purifying thrombomodulin, wherein affinity column chromatography as the main purification step is followed by strong anion exchange chromatography (Patent document 13).